Thermal limits of reptiles - Ecological and environmental constraints on the thermal biology of Malagasy lizards

Thermal limits of reptiles

Ecological and environmental constraints

on the thermal biology of Malagasy lizards

Dissertation with the aim of achieving a doctoral degree at the University of Hamburg

Faculty of Mathematics, Informatics and Natural Sciences Department of Biology

Submitted by Ole Theisinger Hamburg, 2016

Date of oral defense: 13th of January 2017 Dissertation supervisor:

Prof. Dr. Kathrin Dausmann, University of Hamburg Dissertation reviewers:

Prof. Dr. Kathrin Dausmann, University of Hamburg Prof. Dr. Jörg Ganzhorn, University of Hamburg

List of Figures I Summary 1 Zusammenfassung 5

Introduction

Chapter 1

Compensation of thermal constraints along a

natural environmental gradient in a Malagasy

iguanid lizard (Oplurus quadrimaculatus)

Abstract 27 Keywords 28 Introduction 31 Methods 30 Study sites 30 Study species 30

Operative environmental temperature 31

Skin temperature patterns 31

Metabolic measurements 32

Daytime field resting costs 33

Results 33 Discussion 36 Acknowledgements 40 References 40 Supplementary material 47 Author contribution 48

Chapter 2

Behavioural capacity of a heliothermic lizard

(Oplurus saxicola) to compensate for differences

in the thermal environment

Abstract 51 Keywords 52 Introduction 52 Methods 54 Study site 54 Study species 54 Activity transects 55 Focal observations 55

Operative environmental temperature 56

Data analysis 57 Results 57 Discussion 62 Conclusion 65 Acknowledgements 65 References 66 Author contribution 72

Chapter 3

Ecological constraints in the thermal biology of

heliothermic lizards

Operative environmental temperature 79

Temperature profiles 79

Results 80

Operative environmental temperature 80

Temperature profiles and behaviour 80

Discussion 83

Acknowledgements 87

References 87

Supplementary material 92

Discussion

Thermal conditions at the study site 95

Precision and accuracy of thermoregulation in Oplurus spp. 96

Coexistence of Oplurus spp.: similarities and differences 97

Thermal restrictions and energy budgets 98

The value of a shift in Tpref 99

Thermal biology of Zonosaurus laticaudatus 100

Vulnerability to environmental change of Zonosaurus laticaudatus 101

Metabolic acclimatization 102

Implications of body temperature on lizards’ performance 103

Potential mechanisms to compensate high refuge temperature 103

Alternative energy saving strategies 104

Temperature sensitivity of embryonic development 105

Importance of “thermal reality” 106

Importance of thermal biology for conservation and extinction risk 107

Conclusion

References 110

Acknowledgements 119

English language certificate 121

I

List of Figures

Figure 0.1: Hypothetical performance curve of an ectotherm as a function of body temperature (modified from Sinclair et al. 2016). w (Tb): relative

fitness/performance. 11

Figure 0.2: Photographs of study sites in the Andohahela National Park: A) spiny forest; B) gallery forest; C) transitional forest; D) rain forest (photos by Ole Theisinger).

14 Figure 0.3: Map of our study sites. Differently coloured areas show different vegetation

formations. Yellow: spiny forest; black (along rivers): gallery forest; shaded: transitional forest; green: rain forest; white: savannah. Red asterisks show our study sites (modified from Rakotondranary et al. 2011). 15 Figure 0.4: Study species: A) Oplurus quadrimaculatus with an attached temperature

logger; B) Oplurus saxicola in “high” posture with minimal surface contact to the hot rock; C) measuring skin temperature of Zonosaurus laticaudatus by using an infrared thermometer (photos by Wiebke Berg and Ole Theisinger). 16 Figure 1.1: Daily skin temperature (Tskin) profiles of Oplurus quadrimaculatus across an

environmental gradient in southeast Madagascar. The solid line shows Tskin of the lizard and the dashed line shows ambient temperature. The lizard 1) leaves its warm crevice and cools down to ambient temperature before heating up in the sun by basking, 2) is active, 3) cools down with ambient temperature and enters its crevice. Horizontal black bars indicate the scotophase. 34 Figure 1.2: Mean activity skin temperature (Tskin; open circles), mean day Tskin during the

photopase (between 0600 hours and 1800 hours) including periods of inactivity (grey squares), and night Tskin (black triangles) of Oplurus quadrimaculatus in different habitats along an environmental gradient in southeast Madagascar. Error-bars show 95% confidence intervals and lowercase letters (a, b, c and x, y, z) indicate significant differences between habitats. 34 Figure 1.3: Mean operative environmental temperature of Oplurus quadrimaculatus in

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measured with copper models during the same time period. The straight black line shows mean activity skin temperature in each habitat and straight grey lines are standard deviation. Horizontal black bars indicate the scotophase. 35 Figure 1.4: A) Mean daily activity time (DAT) and B) daytime field resting costs (FRC)

of Oplurus quadrimaculatus in different habitats along an environmental gradient in southeast Madagascar. Error-bars show 95% confidence intervals and lowercase letters indicate statistical significance. 36 Figure 1.5: Temperature-field resting metabolic rate (field RMR) relationship for Oplurus

quadrimaculatus. Data points represent repeated measurements from all

individuals and the black line shows the fitted line of the linear mixed effects model (log(field RMR) = 0.09(Tskin) – 5.37) that accounts for an unequal number of data points and repeated measurements. 37 Figure S1.1: Individually marked Oplurus quadrimaculatus in its natural habitat in

southeastern Madagascar with attached temperature logger (photo by Wiebke

Berg). 47

Figure 2.1: Body postures of Oplurus saxicola: A) flat on the ground with maximal surface contact; B) normal in upright position with tail and rear touching the ground; C) high with spread legs and minimal surface contact. 56 Figure 2.2: Minimum (Tmin), mean (Tmean) and maximum (Tmax) ambient temperature

between 0600 hours and 1800 hours in the spiny forest (closed circles) and gallery forest (open squares). Asterisks indicate level of significance (*p < 0.05; **p <

0.001). 58

Figure 2.3: Mean body temperature during activity (left) and single body temperature data over the course of the day (right) of Oplurus saxicola in the spiny forest (closed circles) and gallery forest (open squares). Lines are loess curves for data from the spiny forest (solid line) and gallery forest (dotted line). 58 Figure 2.4: Operative environmental temperature of Oplurus saxicola in the spiny forest

(left) and gallery forest (right). Each differently shaded line represents five-days-average temperatures of a copper model placed in different microhabitats

III including full sun, full shade and crevice. The dashed lines show the activity body

temperature range. 59

Figure 2.5: Different activities of Oplurus saxicola in the course of the day in the spiny forest and the gallery forest. A) Overall activity of individuals in each population, B) shuttling frequency between microhabitats, C) posture change frequency and D) the number of successful feeding events (small circles and squares) and the total number of feeding attempts (big circles and squares). Asterisks indicate level of significance (*p < 0.05; **p < 0.001). 61 Figure 2.6: Overall activity and microhabitat use of Oplurus saxicola in two different

habitats over the course of the day. 62 Figure 3.1: An adult Oplurus quadrimaculatus (top) and a subadult Zonosaurus

laticaudatus are sharing a basking spot on a rock in southeast Madagascar. 78

Figure 3.2: Operative environmental temperature (Te) ranges of Oplurus quadrimaculatus (solid line) and Zonosaurus laticaudatus (dashed line) over the course of the day. Upper and lower lines show maximum Te and minimum Te. Grey bars indicate the central 50% of the activity skin temperature range (dark grey: O.

quadrimaculatus; light grey: Z. laticaudatus). 81

Figure 3.3: Typical skin temperature profiles (solid line) of A) Oplurus quadrimaculatus and B) Zonosaurus laticaudatus and ambient temperature (dashed line) on sunny, cloudless days with a broad operative environmental temperature range. Both species leave their warm crevices after sunrise and cool down with ambient temperature before they heat up through basking in the sun. Oplurus

quadrimaculatus is active at high skin temperature until it cools down with

ambient temperature at sunset and reheats when entering the warm crevice.

Zonosaurus laticaudatus is active in shady leaf litter and crevices. It cools down

directly after a short period of basking in the morning. 81 Figure 3.4: Mean skin temperature of Oplurus quadrimaculatus (n = 48; filled circles)

and Zonosaurus laticaudatus (n = 25; open squares) over the course of the day. 82

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Figure 3.5: Histogram of the relative frequency of activity skin temperature for Oplurus

quadrimaculatus (black bars) and Zonosaurus laticaudatus (white bars). 83

Figure S3.1: Skin temperature profile (solid line) of Oplurus quadrimaculatus. The dashed line shows ambient temperature. The arrow indicates the clearly discernible moment when the temperature logger detached from the lizard. 92 Figure 4.1: Mean number of active individuals of Oplurus saxicola over the course of the

day in different habitats. Solid line: spiny forest; dashed line: gallery forest; dotted

line: transitional forest. 96

Figure 4.2: Prey items of Zonosaurus laticaudatus. Left: Z. laticaudatus is feeding on a hissing cockroach. Right: a millipede partially eaten by Z. laticaudatus. 101 Figure 4.3: Specifically designed copper models for (from left to right) Oplurus saxicola,

O. quadrimaculatus and Zonosaurus laticaudatus to measure the operative

environmental temperature. The hind legs are missing in the models for O.

saxicola due to better correlation with live lizards in heat conductance (photo by

1

Summary

Ectotherms are particularly affected by fluctuations and changes in environmental conditions because their body temperature largely depends on heat exchange with the environment. However, a constant body temperature has the advantage that physiological body functions and performance can be optimized at this temperature. Ectotherms evolved sophisticated mechanisms to either maintain a rather constant body temperature (behavioural plasticity) or to compensate temperature fluctuations through shifts in the thermal reaction norm of physiological processes (physiological plasticity). However, under natural conditions, body temperature is also affected by multiple ecological factors that constrain the thermal scope of an animal.

This dissertation examines the question how ecological requirements and environmental constraints affect the thermoregulation of lizards. I investigated the activity temperature, activity time, thermoregulatory behaviour and the energy budget of three syntopic lizard species (Oplurus quadrimaculatus, O. saxicola and Zonosaurus laticaudatus) from southeast Madagascar. The study took place in the Andohahela National Park, which comprises a unique and steep environmental gradient in temperature and precipitation. Habitats change within a distance of less than 10 km from cool and humid rainforest to hot and dry spiny forest, with moderately tempered gallery and transitional forest in between. The proximity of these extremes and the fact that my study species occur all along the gradient provide an ideal natural setup to investigate the effects of different thermal environments on the thermoregulation of lizards.

In the first chapter, I used temperature loggers to analyse skin temperature patterns of O.

quadrimaculatus in differing habitats along the gradient. The results show a precisely

regulated skin temperature during activity along the entire gradient despite marked differences in the thermal environment. However, cooler and shadier conditions in the rainforest reduced the activity time of individuals by 35%. Additionally, I measured the field resting metabolic rate using open flow respirometry to quantify daytime field resting costs (maintenance costs of non-fasting animals) of the lizards, which were reduced by 28% in the cooler habitat. Hence, in the rainforest, these lizards have less time available for foraging but they also require less food to cover their basic energetic costs.

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Nevertheless, the cool rather than the hot environment seems to present a thermal limitation in this species, because it also matches its distribution limit.

In the second chapter, behavioural observations and temperature measurements of O.

saxicola in the hot spiny forest and the moderately tempered gallery forest reveal how the

activity of this species is adapted to different thermal environments. Individuals from the spiny forest show a bimodal distribution of activity with the highest peak in the morning, reduced activity during midday and again increased activity in the afternoon. In contrast, individuals in the gallery forest show highest activity during midday and activity in the afternoon is constrained by a lack of direct sunlight. Nevertheless, the mean activity body temperature of O. saxicola did not differ between habitats which argues strongly against a change in the temperature set-point and thus against physiological adjustments. This species rather relies on behavioural thermoregulation and compensates thermal differences mainly through shuttling between microclimates and posture changes.

The third chapter compares skin temperature patterns over the course of the day in O.

quadrimaculatus and Z. laticaudatus. While O. quadrimaculatus maintains a permanently

high activity temperature during the day, Z. laticaudatus shows a different but distinct temperature pattern. After elevated skin temperature in the morning during basking, the skin temperature decreases and remains low for the rest of the day during foraging in shady leaf litter and crevices. Despite the opportunity to reheat in the sun, which would improve its performance, Z. laticaudatus remained cool. The elevated skin temperature in the morning most likely promotes physiological processes, such as digestion, detoxification, and immune response. The lower skin temperature thereafter nevertheless allows sufficient performance to forage on snails, hissing cockroaches, millipedes and carrion. While O. quadrimaculatus forages at high body temperature and can therefore satisfy physiological requirements during normal activity, Z. laticaudatus is forced into a trade-off between foraging in cooler microclimates and the need to accelerate physiological processes through elevated body temperature. This dilemma is solved by a temporal and spatial separation of daily activities that is so far unknown in diurnal lizards. This pattern allows for an increase in foraging time and the lizards can exploit food sources further away from the basking site.

Overall, this study shows that behavioural thermoregulation is the major compensatory mechanism for environmental differences in heliothermic lizards under natural

3 conditions. The degree to which the thermal niche is affected by ecological constraints and habitat use varies between species. Oplurus spp. for example, are relatively unaffected by ecological factors because they are able to maintain permanently stable body temperatures through simultaneous foraging and thermoregulation. In contrast, if a species is not able to satisfy ecological and physiological needs simultaneously, because the temperature of the foraging sites is too low, it faces a dilemma. Zonosaurus

laticaudatus solves this trade-off with an unexpected but reasonable split of the day

instead of constant shuttling.

The results of my study are important to understand thermal strategies under natural conditions. This interdisciplinary approach with physiological measures in an ecological context highlights the importance of thermal reality of lizards in the wild. The combination of ecological constraints, thermal strategies and compensatory mechanisms has been largely overlooked because it requires studying animals in their natural environment under natural conditions. It might, however, be exactly this interplay that decides about the future distribution of species and their resilience to habitat modification and climate warming.

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Zusammenfassung

Ektotherme Tiere sind besonders stark von Umweltveränderungen und Fluktuationen der Umgebungstemperatur betroffen, da ihre Körpertemperatur fast ausschließlich durch Wärmeaustausch mit der Umgebung reguliert wird. Da physiologische Prozesse und somit die Leistungsfähigkeit allerdings für eine bestimmte Temperatur optimiert sind, ist eine konstante Körpertemperatur klar von Vorteil. Deshalb nutzen viele ektotherme Tiere hochentwickelte Mechanismen (Thermoregulationsverhalten und/oder physiologische Anpassungen), um Veränderungen in der Umgebungstemperatur zu kompensieren bzw., um die spezifische Körpertemperatur im optimalen Bereich für die Leistungsfähigkeit zu halten. Allerdings wird die Körpertemperatur auch durch ökologische Faktoren beeinflusst, was den verfügbaren Temperaturbereich teilweise deutlich einschränkt.

In dieser Dissertation untersuche ich die Frage, auf welche Weise ökologische und abiotische Faktoren die Thermoregulation von Reptilien beeinflussen. Hierfür habe ich den Effekt von unterschiedlichen Habitattemperaturen auf die Aktivitätstemperatur, die Aktivitätszeiten, das Thermoregulationsverhalten und das Energiebudget von drei syntopen Echsenarten (Oplurus quadrimaculatus, O. saxicola und Zonosaurus

laticaudatus) aus Südostmadagaskar untersucht. Das Untersuchungsgebiet im

Andohahela Nationalpark bietet einen einzigartigen, natürlichen Umweltgradienten bei dem sich auf einer Distanz von weniger als 10 km ein Wechsel vom kühlen, immergrünen Regenwald, über den moderat temperierten Übergangs- und Galeriewald, bis hin zum heißen, trockenen Dornenwald vollzieht. Durch die räumliche Nähe dieser Extreme und die Tatsache, dass die untersuchten Echsenarten entlang des gesamten Gradienten vorkommen, bietet Andohahela ideale Voraussetzungen, um den Einfluss unterschiedlicher Habitate auf die Thermoregulation von Reptilien unter natürlichen Bedingungen zu untersuchen.

Im ersten Kapitel habe ich Hauttemperaturmuster analysiert, die von auf den Tieren befestigten, temperatursensitiven Datenloggern aufgezeichnet wurden. Oplurus

quadrimaculatus thermoregulierte während der Aktivitätszeit, unabhängig von der

Habitattemperatur, sehr präzise und konnte dabei durchgehend eine hohe Hauttemperatur aufrechterhalten. Allerdings gab es im kühlen, schattigeren Regenwald starke Einschränkungen in der täglichen Aktivitätszeit von etwa 35% im Gegensatz zum

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Dornenwald. Gleichzeitig haben respiratorische Messungen und der daraus berechnete Energiebedarf gezeigt, dass die energetischen Kosten um etwa 28% geringer waren. Den Tieren steht demnach in kühleren Habitaten zwar weniger Zeit für die Nahrungssuche zur Verfügung, sie benötigen allerdings auch weniger Nahrung um den niedrigeren Energiebedarf zu decken. In diesem Fall limitiert nicht das warme Habitat (Dornenwald), sondern das kühle Regenwald-Habitat die Tiere, das gleichzeitig auch die Verbreitungsgrenze dieser Art darstellt.

Im zweiten Kapitel zeige ich anhand von Verhaltensbeobachtungen und Körpertemperaturmessungen an O. saxicola im heißen Dornenwald und im gemäßigten Galeriewald, dass die Echsen ihre Aktivitätszeiten den thermischen Gegebenheiten der Habitate anpassen. Die Individuen im Dornenwald zeigten eine bimodale Verteilung der Aktivitätszeiten, mit dem Aktivitätshöhepunkt am Morgen, gefolgt von einem Mittagstief und einer wieder erhöhten Aktivität am Nachmittag. Die Individuen im Galeriewald hingegen zeigten die höchste Aktivität während der Mittagszeit und sind nachmittags durch mangelnde direkte Sonneneinstrahlung stark eingeschränkt. Der Wechsel zwischen verschiedenen Mikroklimata, aber auch die Veränderung der Körperhaltung sind die wichtigsten Thermoregulationsmechanismen bei dieser Art. Die mittlere Körpertemperatur während der Aktivitätszeit zeigte dabei keine Unterschiede zwischen den Populationen, was darauf hindeutet, dass der Sollwert für die Körpertemperatur unverändert ist und es dahingehend keine physiologischen Anpassungen an die Habitate gibt. Insgesamt ist es O. saxicola möglich, durch eine Verschiebung der Aktivitätszeiten und durch die Anpassung der Wechsel zwischen Mikroklimata, Habitatunterschiede auszugleichen.

Im dritten Kapitel vergleiche ich die Hauttemperaturmuster von O. quadrimaculatus und

Z. laticaudatus im Tagesverlauf. Während O. quadrimaculatus eine konstant hohe

Aktivitätstemperatur hält, zeigt Z. laticaudatus einen abweichenden, aber ebenfalls sehr ausgeprägten Hauttemperaturverlauf. Nach einer erhöhten Temperatur während des Sonnens am Morgen sinkt die Hauttemperatur während der Nahrungssuche in der schattigen Laubstreu und in Felsspalten deutlich ab und verbleibt für den Rest des Tages auf diesem deutlich niedrigeren Niveau. Trotz der Möglichkeit, sich wieder in der Sonne aufzuheizen, was eine höhere Leistungsfähigkeit für die Nahrungssuche bedeuten würde, nutzen die Echsen dies nicht. Die erhöhte Körpertemperatur dient vermutlich dazu,

7 physiologische Prozesse, wie Verdauung, Entgiftung oder Immunreaktionen, zu beschleunigen. Die niedrigere Körpertemperatur ermöglicht offensichtlich ausreichende Beweglichkeit bei der Nahrungssuche nach Schnecken, Fauchschaben, Tausendfüßern und Aas. Oplurus quadrimaculatus jagt ohnehin bei einer hohen Körpertemperatur, was gleichzeitig auch die physiologischen Bedürfnisse befriedigt. Hingegen muss Z.

laticaudatus auf der einen Seite physiologische Prozesse durch aktive Thermoregulation

in der Sonne verbessern, auf der anderen Seite findet die Echse seine Nahrung in kühleren Mikrohabitaten. Dieses Dilemma wird durch eine räumliche und zeitliche Trennung dieser Verhaltensweisen gelöst. Eine solch klare Zweiteilung der Hauttemperatur im Tagesverlauf, trotz der Möglichkeit sich wieder aufzuheizen, war bisher nicht bekannt. Durch dieses Muster steht den Echsen mehr Zeit für die Futtersuche zur Verfügung und sie können Nahrungsquellen nutzen, die weiter von den Sonnenplätzen entfernt sind. Insgesamt zeigen meine Ergebnisse, dass Echsen unter natürlichen Bedingungen allein durch Verhaltensanpassungen große Temperaturunterschiede ausgleichen können. Zudem ist das Ausmaß, mit dem die thermische Nische durch ökologische Einschränkungen und die Habitatnutzung beeinflusst wird, artabhängig. Die Oplurus-Arten, zum Beispiel, werden weniger durch ökologische Faktoren beeinflusst, da sie den ganzen Tag über präzise thermoregulieren und gleichzeitig ihre Nahrung suchen. Wenn sich physiologische und ökologische Bedürfnisse aber unterscheiden, kommt es zu einem Konflikt, der, wie im Fall von Z. laticaudatus, auf unerwartete, aber durchaus sinnvolle Weise, durch eine zeitliche und räumliche Aufteilung gelöst werden kann.

Die Ergebnisse meiner Dissertation sind wichtig, um die Kompensationsmöglichkeiten ektothermer Tiere unter natürlichen Bedingungen zu verstehen. Meine interdisziplinäre Studie bringt physiologische Messungen in einen ökologischen Kontext, um die thermische Realität aufzuzeigen, denen die Tiere in der freien Wildbahn ausgesetzt sind. Die Kombination von ökologischen Einschränkungen, thermischen Strategien und Kompensationsmöglichkeiten wurde bisher wenig untersucht, da solche Feldstudien sehr arbeits- und zeitaufwändig sind. Nichtsdestotrotz ist es genau dieses Zusammenspiel verschiedener ökologischer und abiotischer Faktoren, das über die zukünftige Verbreitung von Arten und deren Widerstandsfähigkeit gegen Habitatveränderungen und Klimawandel entscheidet.

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Introduction

Changes in environmental conditions and fluctuations in ambient temperature (Ta) have various and complex effects on the behaviour, ecology and physiology of species (Lillywhite 2016). However, animals must maintain a balanced energy budget over time with a certain body temperature (Tb) that is optimal for physiological functioning. Tb affects biochemical processes, energy expenditure and locomotion performance of an organism (Schmidt-Nielsen 1997; Heldmaier et al. 2013) and fluctuations in Tb disturb the efficiency of these functions (Huey 1982). Ectotherms are especially affected by environmental changes as their Tb depends on heat transfer with the environment (Little and Seebacher 2016) and precision in thermoregulation is a key for optimal performance (Bennett and Ruben 1979; Clarke and Pörtner 2010). Hence, in changing environments compensatory mechanisms are needed to control Tb or to adjust biochemical processes in order to maintain balanced energy budgets.

Ectotherms with relatively high Tb generally outperform species with lower Tb, which is also known as the “hotter-is-better” hypothesis (Angilletta et al. 2010). However, in contrast to endotherms, few ectotherms produce a significant magnitude of body heat. So far, only a few fish species, such as tuna, lamnid sharks (Carey and Teal 1968; Carey et

al. 1971) and the deep sea opah (Wegner et al. 2015), are known to attain permanently

higher Tb than the ambient water temperature. Furthermore, pythons and tegu lizards show temporary endothermy through shivering thermogenesis but only during reproduction (Harlow and Grigg 1984; Tattersall et al. 2016). Most ectotherms thus depend on their thermal environment for achieving suitable Tb. Gaining body heat is not always the major concern for species though. Quite the contrary, the challenge for animals in hot environments is staying cool because if Ta exceeds the critical maximum Tb, animals face overheating and death (Kearney et al. 2009).

A high Tb is generally beneficial for physiological performance but the stability of Tb (avoiding Tb that would be too cold and too hot) is even more important because physiological function peaks at a certain Tb (Huey and Slatkin 1976). This is less challenging in large ectotherms, for example in the Komodo dragon Varanus

komodoensis, because high body mass and a small surface-to-volume ratio facilitate

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In contrast, the greater surface to volume ratio in smaller reptile species results in increased heat conduction and faster heating and cooling rates, and thus a limited potential to store attained body heat (Stevenson 1985).

The concept that ectotherms are able to control their Tb and that temperature is an essential factor in reptile ecology and physiology has been developed in several pioneering studies. Mosauer (1936) experimentally showed that the desert lizard Uma

notata does not tolerate high sand temperatures of more than 55°C, even though it

inhabits this harsh environment. Cowles and Bogert (1944) showed in a comprehensive ecophysiological study that “cold-blooded” organisms do not just passively take on the temperature of their environment. Instead, they have sophisticated behavioural mechanisms that allow them to precisely regulate their Tb within a wide range of ambient conditions. Colbert et al. (1946) studied the heat tolerance of the American alligator

Alligator mississippiensis and revealed the important effect of posture and body

orientation on thermoregulation. Bogert (1949) demonstrated that behavioural thermoregulation of spiny lizards (Sceloporus spp.) can be surprisingly precise with Tb fluctuations of less than 3°C during activity. Norris (1953) recognized that Tb, Ta and habitat structure are important factors for a detailed description of the ecology of the desert iguana Dipsosaurus dorsalis. Finally, Huey and Slatkin (1976) and Huey and Stevenson (1979) integrated thermal physiology and ecology of ectotherms. By reviewing analytical methods of describing and comparing aspects of performance, they created a hypothetical performance curve of ectotherms that is still used to describe temperature related body functions until today (Fig. 0.1).

This performance curve is essential for an understanding of the thermal reaction norm. No matter which physiological trait is selected (e.g. endurance, sprint speed, hearing, or digestion), all show similar performance patterns and this may even be transferable to endotherms (Huey and Kingsolver 1989; Boyles et al. 2011). The lower critical Tb is the lowest Tb tolerated by an ectotherm before death. Performance then increases with increasing Tb until it reaches the maximum (thermal optimum, Topt). The curve has a negative skew and drops sharply towards the upper critical Tb. For this reason, the preferred body temperature (Tpref; selected Tb by an undisturbed ectotherm in an artificial temperature gradient; Huey and Kingsolver 1989) and activity Tb in the wild are often

11 below but closely related to Topt to maintain a high performance level with an additional safety margin to the lethal critical maximum (Huey et al. 2012).

Figure 0.1: Hypothetical performance curve of an ectotherm as a function of body temperature (modified from Sinclair et al. 2016). w (Tb): relative fitness/performance.

The fact that lizards are able to achieve Tb that deviate from Ta shows that Ta itself does not adequately describe the thermal reality for these animals. The operative environmental temperature range (Te; theoretically obtainable Tb range for a species in a given habitat) is therefore used to describe the thermal scope of an animal across microhabitats rather than a single record of Ta (Bakken et al. 1985; Bakken 1992; Dzialowski 2005). Lizards are able to use a range of environmental temperatures depending on their capacity of behavioural thermoregulation. The magnitude of the Te range depends on the thermal heterogeneity of the environment, temporal and spatial habitat use of the species and its body mass, shape and colouration (Bakken and Angilletta 2014; Sears and Angilletta 2015). Hence, Tb can be stable despite large environmental differences and even if Ta fluctuates, Te can still provide a constantly suitable range of temperatures.

If Tb deviates from Topt or it is outside the thermal reaction norm, animals have two options to retrieve optimal performance. Either they actively select sites with optimal

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thermal conditions (behavioural thermoregulation) or the optimal range of the thermal reaction norm shifts to match the current Tb (physiological acclimatization) (Glanville and Seebacher 2006). These two strategies have fundamentally different approaches but they are not necessarily mutually exclusive.

The great advantage of behavioural thermoregulation is that responses are quick, flexible, and, depending on the animals’ ability and the environment, highly efficient (Munoz et al. 2014). The disadvantage is that behavioural thermoregulation can only occur within the thermal scope of Te. Besides adjustments of activity time, posture, orientation to the sun, shuttling and panting are the most common behavioural traits that are used by terrestrial ectotherms for thermoregulation (Grant and Dunham 1988; McConnachie et al. 2009; Gifford et al. 2012). Physiological adjustments are more inert but they optimize biochemical processes if changes in temperature are short term relative to the animal’s life-span (Angilletta et al. 2002). Based on changes in regulatory enzymes, a shift of the thermal reaction norm allows enhanced performance at otherwise suboptimal Tb. This strategy is used by many reptiles to compensate seasonal changes in the environment but short-term changes of ambient conditions, e.g. severe weather events over several weeks, may also provoke physiological adjustments (Seebacher 2005). Under natural conditions, behavioural and physiological plasticity often act jointly. The plastic modification of Tpref, for example, has been proposed to reduce costs for thermoregulation and contributes to buffer unsuitable conditions (Gvozdik 2012) and a seasonal change of Tpref in newts does indeed increase their time window for thermoregulation (Hadamova and Gvozdik 2011). Ectotherms that select lower Tb show changes in behaviour (Tb selection) accompanied by changes in the thermal reaction norm (lower set-point of Tb). Hence, thermoregulatory behaviour and physiological acclimatization do not represent mutually exclusive thermal responses. They have coevolved, in at least some species, to help offset potential costs associated with each strategy (Little and Seebacher 2016).

At the same time, behaviour and physiology are often directly affected by biotic and abiotic influences (Clusella-Trullas and Chown 2014). Although many lizards aim to achieve Topt in the wild, their actual activity Tb often deviates from the expected one (Huey et al. 1989). Predation pressure (Herczeg et al. 2008), nutritional state and food quantity (Gatten 1974; Gienger et al. 2013), nocturnal activity (Huey et al. 1989), competition (Downes and Shine 1998), reproductive state (Harlow and Grigg 1984) and

13 the lack of suitable microclimates (Huey and Slatkin 1976; Stellatelli et al. 2013) force animals into trade-offs between physiological and ecological demands. For this reason, results from semi-natural field experiments and observations from free-ranging animals can differ significantly (Clusella-Trullas and Chown 2014). For example, Tpref of two populations of the New Zealand common gecko Hoplodactylus maculatus differed in field acclimatized individuals but not after acclimation in the laboratory (Tocher 1992). The legless lizard Lialis burtonis shows post-feeding thermophily in the laboratory but not in semi-natural enclosures (Wall and Shine 2008) and the arboreal banded snake

Hoplocephalus stephensii selects lower Tb in the wild than it does in an artificial

temperature gradient (Fitzgerald et al. 2003). However, these differences between the realized and the fundamental thermal niche are important because these data are often used for the prediction of animals’ future distribution.

Mechanistic models on the (local) extinction of species in several taxa are often based on physiological tolerances measured under controlled laboratory conditions with limited reference to the realized ecological niche to predict the effect of environmental change (Sinervo et al. 2010; Böhm et al. 2016). On the one hand, lizards are constrained in their Tb selection by these multiple ecological factors. On the other hand, lizards may have more options for thermoregulation (e.g. burrowing, cooling in water) than laboratory studies would reveal and hence, more potential to buffer environmental fluctuations than expected (Wall and Shine 2008). However, different species are affected in different ways and empirical studies on behavioural and physiological compensation under natural conditions are needed to understand the magnitude of species resilience (Basson and Clusella-Trullas 2015; Pacifici et al. 2015).

Madagascar, as one of the world’s hottest biodiversity hotspots, is known for its high species richness and the high level of endemism but also for a high level of degradation (Myers et al. 2000; Ganzhorn et al. 2001; Harper et al. 2007). Additionally, the climate is hypervariable, unpredictable and the predicted temperature increase affects particularly the dry southern part of the island (Dewar and Richard 2007; Tadross et al. 2008; Hannah

et al. 2008). In the southeast, the Anosy Mountains act as a rain barrier for humid winds

from the Indian Ocean and create a steep environmental gradient on the western flank. While the mountains consist of evergreen rainforest, less than 10 km further west spiny forest is the predominant vegetation form that continues all the way to the southwest.

14

Precipitation decreases from more than 2,400 mm/year to 400 mm/year and mean Ta increases approximately 8°C within this short distance (Goodman 1999; Rakotondranary

et al. 2011). This gradient has led to high habitat diversity on a small geographic scale

(Fig. 0.2). The extremes (spiny and rain forest) are connected by patches of moderately tempered transitional forest (Fig. 0.3). While rain forest and spiny forest are protected in the Andohahela National Park, the transitional forest lies between the protected areas and is rather degraded and fragmented. Within the spiny forest parcel, stretches of gallery forest can be found along rivers. This forest type is cooled by the river and also moderately tempered, similar to the transitional forest. The gallery forest is a very heterogeneous habitat with large, evergreen trees and, in parts, large rocks or sandy spots and open water is available year-round. The gallery forest is often not wider than 100 m but it is an important refuge for animals within the hot and arid landscape (Mares and Ernest 1995). The proximity of these habitats with differing thermal conditions and the fact that these habitats share many of their reptile species (Theisinger 2009) provide an excellent setup for the investigation of compensatory thermoregulation mechanisms.

Figure 0.2: Photographs of study sites in the Andohahela National Park: A) spiny forest; B) gallery forest; C) transitional forest; D) rain forest (photos by Ole Theisinger).

15

Figure 0.3: Map of our study sites. Differently coloured areas show different vegetation formations. Yellow: spiny forest; black (along rivers): gallery forest; shaded: transitional forest; green: rain forest; white: savannah. Red asterisks show our study sites (modified from Rakotondranary et al. 2011).

The overall goal for this dissertation was to investigate lizards’ behavioural and physiological capacity to compensate for differences in the thermal environment and to assess the effect of foraging activity on their thermal ecology. Furthermore, we aimed to evaluate environmental and ecological effects on the energy budgets of lizards. We used three lizard species as model organisms (Oplurus quadrimaculatus, O. saxicola and

Zonosaurus quadrimaculatus; Fig. 0.4) which occur sympatrically all along the steep

temperature and precipitation gradient in Andohahela (Glaw and Vences 2007). Field work was conducted between October 2010 and April 2012. All species are diurnal and occur in relatively high densities. Moreover, our study species, and particularly O.

saxicola and O. quadrimaculatus, are highly philopatric even during their daily activity,

16

Figure 0.4: Study species: A) Oplurus quadrimaculatus with an attached temperature logger; B) Oplurus

saxicola in “high” posture with minimal surface contact to the hot rock; C) measuring skin temperature of Zonosaurus laticaudatus by using an infrared thermometer (photos by Wiebke Berg and Ole Theisinger).

The dissertation consists of three research projects to answer the following questions: 1. The combination of behaviour and physiology for thermoregulation and a

balanced energy budget is crucial to understand an animal’s potential to compensate environmental fluctuations and to persist in the face of climate change (Basson and Clusella-Trullas 2015). To investigate whether a decline in Te along the environmental gradient in southeastern Madagascar is compensated through lowered activity Tb or through changes in activity time, we studied intraspecific differences in the daily skin temperature (Tskin) pattern (as proxy for Tb; Berg et

al. 2015) of O. quadrimaculatus. Additionally, we measured the field resting

metabolic rate (field RMR) and used the temperature-field RMR regression to estimate field resting costs in free ranging individuals. A comparison of these

17 costs among habitats provides insight into possible energetic advantages of colder versus warmer environments.

 Does O. quadrimaculatus make use of physiological adjustments or are differences in the thermal environment solely compensated behaviourally?  Do field resting costs of O. quadrimaculatus differ between the habitats?

2. Unfavourable Tb causes changes in behaviour in lizards, because they have to compensate or to avoid extreme environmental temperatures which often conflicts with normal activity (Sun et al. 2001). In hot habitats, this might result in bimodal activity patterns to avoid midday heat (Sinervo et al. 2010). In addition, adjustments in foraging time and thermoregulatory behaviour are necessary to compensate for constraints in activity time. We investigated the direct effect of environmental differences (hot spiny forest and moderate gallery forest) on overall activity, thermoregulatory behaviour and foraging over the course of the day in O.

saxicola through focal observations, scan sampling and Tb measurements.

 Do these lizards show similar activity patterns in both habitats?

 Do these lizards show adjustments in mean Tb to the different thermal environments?

 Does microhabitat use and foraging behaviour differ between the habitats?

3. Ecological factors, such as foraging strategy, often differ between sympatric species to minimize interspecific competition. Trade-offs between physiological demands, such as a relatively constant Tb for optimal performance, and ecological constraints might result in spatial or temporal differences in microhabitat use between sympatric species, which can have a major effect on the thermal niche of the lizards (Clusella-Trullas and Chown 2014; Murray et al. 2016). We used an interspecific comparison to investigate the constraining effect of foraging ecology and microhabitat use on activity Tskin. We compared Tskin patterns of the sympatric lizards O. quadrimaculatus and Z. laticaudatus, which experience the same Te conditions but differ in their foraging ecology (sit-and-wait forager, respectively, active forager).

18

Table 1: Glossary

Glossary

Body temperature (Tb)

Core temperature of a lizard. Measured with thermocouples inserted into the cloaca (approx. 1.5 cm).

Skin temperature (Tskin)

Temperature measured on the back of lizards using either infrared

thermometer or attached temperature logger. It can be used as proxy for Tb in our study species (Berg et al. 2015) Ambient temperature (Ta)

Temperature of the environment measured in full shade in one meter height.

Operative environmental temperature (Te)

Theoretically attainable Tb range for an ectotherm in a given habitat. Measured with a number of specifically designed copper models equipped with

temperature loggers, distributed in the natural environment of a species (Bakken 1992).

Thermal optimum (Topt)

Tb at which performance is maximal for a certain physiological trait (Huey and Stevenson 1979).

Preferred body temperature (Tpref)

Selected Tb of an ectotherm in an artificial temperature gradient without biotic disturbance (Huey and Stevenson 1979).

Lower critical Tb

Lowest Tb tolerated by an ectotherm associated with the loss of righting response and of locomotory functioning (Huey and Stevenson 1979).

Upper critical Tb

Highest Tb tolerated by an ectotherm associated with the loss of righting response and the onset of muscle spasms (Huey and Stevenson 1979). Thermal tolerance

Range between lower critical Tb and upper critical Tb (Huey and Stevenson 1979).

Field resting metabolic rate (field RMR)

Metabolic rate of a resting, non-fasting animal measured across a range of temperatures.

19

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27

Thermal constraints along an environmental gradient

Chapter 1

Compensation of thermal constraints along a

natural environmental gradient in a Malagasy

iguanid lizard (Oplurus quadrimaculatus)

Ole Theisinger, Wiebke Berg & Kathrin H. Dausmann

Dept. of Functional Ecology, Zoological Institute, University of Hamburg, 20146 Hamburg, Germany.

In press at the Journal of Thermal Biology, DOI: 10.1016/j.jtherbio.2017.01.005

Abstract

Physiological or behavioural adjustments are a prerequisite for ectotherms to cope with different thermal environments. One of the world’s steepest environmental gradients in temperature and precipitation can be found in southeastern Madagascar. This unique gradient allowed us to study the compensation of thermal constraints in the heliothermic lizard Oplurus quadrimaculatus on a very small geographic scale. The lizard occurs from hot spiny forest to intermediate gallery and transitional forest to cooler rain forest and we investigated whether these habitat differences are compensated behaviourally or physiologically. To study activity skin temperature (as proxy for body temperature) and the activity time of lizards, we attached temperature loggers to individuals in three different habitats. In addition, we calculated field resting costs from field resting metabolic rate to compare energy expenditure along the environmental gradient. We found no variation in activity skin temperature, despite significant differences in operative environmental temperature among habitats. However, daily activity time and field resting costs were reduced by 35 % and 28 % in the cool rain forest compared to the hot spiny forest. Our study shows that O. quadrimaculatus relies on behavioural mechanisms rather than physiological adjustments to compensate thermal differences between habitats. Furthermore, its foraging activity in open, sun exposed habitats facilitates such a highly effective thermoregulation that cold operative temperature, not energetically expensive heat, presents a greater challenge for these lizards despite living in a hot environment.

28

Thermal constraints along an environmental gradient

Keywords

Behavioural compensation; energy expenditure; metabolic rate; operative environmental temperature; physiological adjustment; reptiles.

Introduction

Species’ distribution limits are defined by various parameters (Guisan and Thuiller 2005). In ectotherms, the distribution of operative environmental temperature (Te), i.e. the range of attainable body temperatures (Tb), is one of the key factors (Angilletta 2009; Huey et

al. 2012; Sinervo et al. 2010). Even efficient and precise thermoregulation, as found in

heliothermic lizards, still depends on Te to provide suitable Tb (Bakken and Angilletta 2014). If Te is too high, lizards must retreat to cool refuges to avoid overheating. If Te is too low, the animals’ performance is reduced. Sinervo et al. (2010), for example, predicted that Mexican lizards are prone to extinction because of increasing ambient temperature and prolonged hours of restriction (i.e. periods of forced inactivity due to unsuitably high ambient temperature). Tropical lizards are thought to show narrow thermal tolerances because they have adapted to relatively stable climates (Ghalambor et

al. 2006; Huey et al. 2009; Janzen 1967). However, recent results contradict this

assumption (Leal and Gunderson 2012). A variety of microclimates, altitudinal gradients, and pronounced seasonality provide a wide range of environmental conditions in the tropics (Dewar and Richard 2007) and thus might have led to higher thermal tolerances than assumed so far.

The prerequisite for ectotherms to occur in differing thermal environments are physiological or behavioural adjustments. Physiological adjustments comprise changes in physiological rates (Seebacher et al. 2015) or shifts in the thermal tolerance of a species (Gunderson and Stillman 2015). Furthermore, the preferred body temperature [Tpref; the selected Tb of undisturbed ectotherms in an artificial temperature gradient (Huey and Kingsolver 1989)] and the physiological optimum can be lowered or increased in accordance with ambient conditions (Blouin-Demers et al. 2000; Clusella-Trullas and Chown 2014). An upshift of the Tb set-point as a response to higher ambient temperature, for example, increases the potential activity time and might reduce costs for thermoregulation in the face of climate change (Gvozdik 2012). Despite Tb selection

29

Thermal constraints along an environmental gradient

being a behavioural response, the driver for this adjustment are changes in the thermal reaction norm and hence of physiological nature (Little and Seebacher 2016). Lizards that are exposed to different temperatures over several weeks in the laboratory show acclimation of Tpref (Blumberg et al. 2002). This effect was also observed under natural conditions between seasons (Diaz et al. 2005; Seebacher and Grigg 1997) but there is no or only little evidence for adjustments of Tpref to different habitats and altitudes (Gvozdik and Castilla 2001; Van Damme et al. 1989). However, this apparent absence of physiological adjustments is not necessarily equivalent to an incapability of lizards to acclimatize.

Other mechanisms, such as changes in thermoregulatory behaviour and adjusted daily activity time, are known to be effective (Gvozdik 2002) and might be preferential to physiological acclimatization under natural circumstances (Gunderson and Stillman 2015). Thermal constraints through high ambient temperature and the effect of climate warming have often been studied (Deutsch et al. 2008; Huey et al. 2009; Kearney et al. 2009; Sinervo et al. 2010). Since metabolic rate increases exponentially with Tb, climate warming might lead to excessive energetic costs (Kearney 2013). These can be assessed by calculating maintenance costs, i.e. basic metabolic costs in fasting and resting ectotherms, using field Tb (or a proxy for field Tb) and the corresponding metabolic rate (Kearney and Porter 2004). However, low ambient temperature can also be challenging, especially for warm-adapted species (Grbac and Bauwens 2001; Van Damme et al. 1987). Lower Tb may be beneficial for reducing energy expenditure (Christian et al. 1996; Huey

et al. 1989) but sprint speed, digestion, nutrient assimilation and other physiological

functions may be negatively affected by suboptimal performance (Angilletta et al. 2002; Huey and Kingsolver 1989).

To study these compensatory mechanisms under natural conditions, we chose one of the world’s steepest environmental gradients, which is found in southeastern Madagascar. The unique gradient in ambient temperature, humidity and rainfall, connects dry spiny forest and humid rain forest via transitional forest and gallery forest along rivers within less than 5 km (Goodman 1999). Due to this very small geographic scale, species could potentially move between all habitats within a lifetime or sooner, depending on mobility, and reptile species from the hot and dry spiny forest can also be found in the cooler rain forest (pers. obs.).

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Thermal constraints along an environmental gradient

Duméril’s Madagascar swift (Oplurus quadrimaculatus) is one of these species. It occurs in a wide range of habitats with strikingly different environmental conditions including the climatic extremes of hot, dry spiny forest and humid, cool rain forest. We analyzed daily skin temperature (Tskin) patterns (as a proxy for Tb; Berg et al. 2015) in individuals across the entire hot to cold environmental gradient including all four habitat types. Our aim was to examine if these lizards compensate thermal differences through adjustments in activity Tskin or through differences in daily activity time. In addition, we measured field resting metabolic rate (field RMR) and compared individuals’ field resting costs (maintenance costs of non-fasting lizards at daytime) to assess the effect of differing thermal environments on basic energy needs.

Methods

Study sites

Our study sites were located in the Andohahela National Park (24°57'S, 46°35'E) on the western slope of the Anosy Mountains in southeast Madagascar. Within our study area, the distance between the two most divergent habitats (spiny forest and rain forest) is less than 5 km. The spiny forest (150 – 160 m a.s.l.) is characterized by scant and xerophile vegetation such as the octopus tree (Dideracea spp.) and the evergreen rain forest (400 – 430 m a.s.l.) consists of large shady trees and dense understory. The transitional forest (280 – 380 m a.s.l.), as the name implies, comprises mixed vegetation with similar moderate environmental conditions as the gallery forest along rivers (130 – 140 m a.s.l.; Andriaharimalala et al. 2011).

Study species

Oplurus quadrimaculatus is a heliothermic iguana with a body mass of (mean ± SD) 76.5

± 10.5 g (N = 310) and a snout-vent-length of 12.8 ± 0.6 cm. It inhabits open rocky habitats and is a sit-and-wait forager that feeds mainly on flying insects. Its main distribution is the spiny forest of southern Madagascar but there are also populations at higher altitudes and more humid habitats (Glaw and Vences 2007). This species is highly philopatric with a small home range of sometimes less than 50 m2. Hence, animals can easily be located for recapture.

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Thermal constraints along an environmental gradient

Operative environmental temperature

We used specifically designed copper models (three models per habitat) that matched the lizards in shape and colour to characterize the thermal habitat of individual O.

quadrimaculatus (Dzialowski 2005). We equipped these models with temperature loggers

(Thermochron iButtons, model DS1921G; resolution ± 0.5°C; weight 3.3 g; Maxim Integrated Products Inc., San Jose, California, USA.; calibrated in a water bath before use). Calibration against live animals showed that filling the copper models with fine sand revealed the best correlation (Pearson correlation coefficient = 0.962; p < 0.001) with no differences in heating and cooling rates between copper models and lizards (t33 = 0.857; p = 0.397). After programming the loggers to record the core temperature of the model every five minutes, we distributed three models in each habitat (full sun, shade and crevice) in order to cover a representative Te-range. Measurements were made for six consecutive days in each habitat on cloudless, sunny days to ensure similar abiotic conditions.

Skin temperature patterns

To measure daily Tskin patterns, we noosed 48 individuals from different sites across the designated habitats during the rainy season (Mid-October to Mid-March) of 2009/2010, 2010/2011 and 2011/2012. We assume a similar sex-ratio in each habitat because this species is highly philopatric with a very small home range and it occurs in pairs. However, the certain identification of sex was not always possible and thus sex is not taken into account. We glued calibrated temperature loggers on the animals’ backs and released them at their points of capture (Fig. S1.1). To make sure that the weight of the device did not exceed the recommended 5 % of the animals’ body weight (Lovegrove 2009), we only equipped adult individuals with a minimum body mass of 70 g with a temperature logger. We used superglue (UHU Sekundenkleber, UHU GmbH, Bühl, Germany) to attach the devices. The recording interval was set to five minutes and the temperature loggers were able to store up to 2084 data points, which led to a maximal measuring time of seven continuous days. We recaptured individuals after five to seven days to remove the loggers. We gained Tskin data from 13 individuals in the dry spiny forest, 25 individuals in the moderate gallery and transitional forest, and 10 individuals in the humid rain fores. Tskin is highly related to Tb and it can be used as a substitute in this species (Berg et al. 2015). In contrast to day Tskin, which includes the entire photoperiod